&lt;P&gt;Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target for degradation regulatory proteins and misfolded proteins. During cell stress, chaperones play an essential role in preventing the appearance of folding intermediates that lead to irreversibly damaged and aggregated proteins. They promote recovery from stress by disaggregating and reactivating proteins, a process not long ago thought to be impossible. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation and misfolding are primary contributors to a large number of human diseases, including Alzheimers, Parkinsons, type II diabetes, cystic fibrosis, and prion diseases. Understanding how chaperones function and how they interact with proteases will provide the foundation for discovering cures and preventions for the devastating diseases caused by protein misfolding, premature degradation, and formation of toxic protein aggregates.&lt;/P&gt;&lt;P&gt;Previously we showed that Escherichia coli ClpA, an AAA+ protein and the regulatory component of ClpAP protease, has molecular chaperone activity. This work and the work of others demonstrated that Clp ATPases comprise a family of ATP-dependent chaperones. Two members of the Clp family, ClpB of prokaryotes and Hsp104 of yeast, have been shown to dissolve aggregated proteins in combination with a second molecular chaperone, DnaK/Hsp70. However, prior to our work ClpB and Hsp104 had not been demonstrated to possess protein remodeling activity in the absence of the DnaK/Hsp70 chaperone system. We have shown that both proteins have the intrinsic ability to disaggregate some aggregates and that the products of disaggregation are unfolded polypeptides. This work supports a mechanism in which polypeptides are extracted from aggregates by forcible unfolding and translocation through the central channel of the ClpB/Hsp104 hexameric ring. The innate disaggregating and unfolding activities of both ClpB and Hsp104 are elicited in vitro by using mixtures of ATP and ATPgammaS (a slowly hydrolyzed and nonphysiological ATP analog). One interpretation is that in the presence of the mixture of nucleotides, substrates can be held (a function requiring ATP binding and supported by ATP&amp;#947;S) and unfolded and translocated (functions requiring ATP hydrolysis). Because ClpB/Hsp104 acts in conjunction with the DnaK/Hsp70 system in vivo, these results suggest that one role of the DnaK/Hsp70 chaperone system may be to synchronize substrate holding and unfolding by ClpB/Hsp104.&lt;/P&gt; &lt;P&gt;Although it has been known for a decade that the ClpB chaperone is able to dissolve aggregates in conjunction with the DnaK chaperone system, the specific roles of ClpB and DnaK in disaggregation are not well understood. To gain insight into the cooperation between chaperones we are using site-directed mutagenesis to map regions of ClpB that are important for collaboration in disaggregation with DnaK. We obtained some mutant ClpB proteins that are functional independent of the DnaK system, similar to wild type ClpB. However, unlike wild type ClpB, they are unable to collaborate with DnaK chaperone system in protein disaggregation. Other mutants are unable to act alone, unlike wild type, but are active in combination with the DnaK system. Thus, we have identified specific ClpB residues and regions that are important for the collaboration between ClpB and DnaK. The further characterization of these mutants will more clearly define the roles of the two chaperone systems.&lt;/P&gt;&lt;P&gt;We have also been exploring the mechanism ATP utilization by ClpB, which is a hexameric protein containing 12 ATP binding sites arranged in two rings of six. Each ClpB protomer contributes one nucleotide-binding site to each ring. Results from subunit mixing experiments show that when ClpB acts alone, approximately six active and six inactive nucleotide-binding sites are required for optimal protein remodeling. The location of the active and inactive sites in the hexamer is not important. Approximately one protomer with two hydrolytically active ATP binding sites per hexamer is sufficient to support remodeling activity, indicating that ClpB can act by a probabilistic mechanism in the absence of the DnaK system. However, when ClpB acts in conjunction with the DnaK system, introduction of approximately one protomer with two inactive ATP binding sites blocks protein disaggregation, supporting a sequential mechanism of ATP utilization by the two rings. Taken together the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary from probabilistic to sequential depending on the presence of the DnaK system and the specific substrate.&lt;/P&gt; &lt;P&gt;We have recently discovered that another Clp/Hsp100 protein, ClpX, participates in cell division of E. coli. ClpX associates with a proteolytic component, ClpP, forming an ATP-dependent protease, ClpXP. In vitro, ClpXP degrades FtsZ, the major cytoskeletal protein in bacteria and a tubulin homolog. FtsZ polymerizes and forms a ring where constriction occurs to divide the cell. Both FtsZ protomers and polymers are degraded by ClpXP;however, polymerized FtsZ is degraded more rapidly than the monomer. Deletion analysis of ClpX showed that the N-terminal domain of ClpX is important for polymer recognition. By deletion analysis of FtsZ, we found that the C-terminus of FtsZ contains a ClpX recognition signal. In vivo, FtsZ is turned over slower in a clpX deletion mutant compared to a wild-type strain. Overexpression of ClpXP results in increased FtsZ degradation and filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers.&lt;/P&gt;